A β -globin Gene, Inactive in the K562 Leukemic Cell, Functions Normally in a Heterologous Expression System

• Published in: Proceedings of the National Academy of Sciences - PNAS 1984-07, Vol.81 (14), p.4485-4489
• Main Authors: Fordis, C. Michael, Anagnou, Nicholas P., Dean, Ann, Nienhuis, Arthur W., Schechter, Alan N.
• Format: Article
• Language: English
• Subjects: Adults; Alleles; Cell Line; Cloning, Molecular; COS cells; DNA; DNA - analysis; DNA Restriction Enzymes - metabolism; Endonucleases - metabolism; Exons; Gene expression; Gene Expression Regulation; Genes; Genetic mutation; Globins - genetics; Humans; K562 cells; Leukemia - genetics; Messenger RNA; RNA; RNA - analysis; Single-Strand Specific DNA and RNA Endonucleases; Transcription, Genetic
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.81.14.4485

The K562 human leukemia cell is an erythroid-like cell that may serve as a model for the study of globin gene expression in transcriptionally active human erythroid cells. K562 cells express all globin genes with the exception of that for β -globin; failure to produce β -globin could result from an acquired mutation in each of the β -globin genes or from an alteration in the regulatory factor environment of the β -globin gene. To uncover a possible acquired mutation, restriction endonuclease analysis of genomic K562 DNA and expression studies of a cloned K562 β -globin gene were carried out. Restriction endonuclease analysis revealed no structural alteration of the K562 β -globin genes. Analysis of the polymorphic Ava II site in intervening sequence 2 of the β -globin gene showed that K562 cells contain two different β -globin alleles, both of which are inactive. A K562 β -globin gene was cloned, ligated into the expression vector pLTN3B, and introduced into COS cells. Transcripts were analyzed by RNA blot, dot blot, S1 nuclease mapping, and primer extension assay. The cloned K562 β -globin gene was transcribed in COS cells as efficiently as a normal β -globin gene introduced into COS cells; the mRNA was 10 S and polyadenylylated; the 5′and 3′termini and the processing of transcripts were identical to that of mRNA transcribed from a normal gene. Based on these data we suggest that the absence of β -globin gene expression results not from an alteration in the β -globin gene, but from a quantitative or qualitative alteration in a trans-acting factor important in β -globin gene expression.